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«Title of Dissertation: STRUCTURAL STUDY OF THE EARLY SECRETORY PROTEIN B AND ATYPICAL RIO2 KINASE Soheila Bahmanjah, Doctor of Philosophy, 2015 ...»

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ABSTRACT

Title of Dissertation: STRUCTURAL STUDY OF THE EARLY

SECRETORY PROTEIN B AND ATYPICAL RIO2

KINASE

Soheila Bahmanjah, Doctor of Philosophy, 2015

Dissertation Directed By: Associate Professor Nicole LaRonde

Department of Chemistry and Biochemistry Mycobacterium tuberculosis, Mtb, is a successful pathogen that secretes variety of proteins to manipulate the host defense mechanisms and create a proper environment for its survival. The progression of the disease depends on the regulated secretion of essential virulence factors such as culture filtrate protein-10 (CFP-10), and early secretory antigenic target-6 (ESAT-6). Mtb regulates the secretion of the virulence factors through interactions of MycP1 protease with EspB (Early secretory protein B) protein.

One of the goals of this thesis was to provide the first crystal structure of EspB protein in order to gain insight into the expression, secretion, function and transmembrane translocation of this protein. EspB292 (residue 1-292) structure has 16 monomers in the asymmetric unit that are arranged into a set of four homotetramers. To clarify the components that affect oligomerization of the EspB292 in the biologically relevant conditions, we set up experiments that mimic the phagosomal environment.

  In addition to work done on EspB protein, structural determination of atypical Chaetomium thermophilum Ct-Rio2 in presence of transition state analogs such as sodium orthovanadate and beryllium fluoride is reported to define the importance of individual domains in binding ATP. Rio2 is required for site D cleavage and maturation of the small subunit ribosomal RNA (rRNA). In this study, the structures of the Ct-Rio2 and its binding to ADP/BeF2 and ADP/VO42- are investigated. The structure are expected to mimic the transition state of the phosphoryl transfer from ATP to Asp257 in Rio2’s active site and the subsequent hydrolysis of the aspartyl phosphate that could power late cytoplasmic 40S subunit biogenesis.

Collaborative work on the structural characterization of the G228 Ct-Rio2 mutant protein is also reported. Our goal is to investigate the evolutionary relationship between Rio kinases and the canonical eukaryotic protein kinases. Our hypothesis is that canonical eukaryotic protein kinases have evolved from Rio kinases. We have made several mutations in a key differentiating position of the kinase domain sequence of Ct-Rio2, G228, and have collected biochemical data to test the ability of the mutated Rio kinase to autophosphorylate and carry out ATP hydrolysis.

 

STRUCTURAL STUDY OF THE EARLY

SECRETORY PROTEIN B AND ATYPICAL RIO2 KINASE

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Advisory Committee:

Professor Nicole LaRonde, Chair Professor Steve Rokita Professor Daniel Falvey Professor Herman O. Sintim Professor Roy Mariuzza, Dean’s Representative   © Copyright by Soheila Bahmanjah   During my PhD program, I have worked in two labs. During my time in Prof. Jeffery T. Davis lab, I started the study of the amphiphilic natural products for the purpose of anion transport across the lipid bilayer of the membrane. For information on this project the reader is referred to the following research article.

Bahmanjah, S., Zhang, N., Davis, J. T. Monoacylglycerols as transmembrane Cl− anion transporters. Chem. Commun. 2012, 48, 4432-4434.

In Dr. Nicole LaRonde’s lab, I worked on three main projects that are described in detail in the next seven chapters.

–  –  –

First and foremost, I would like to dedicate this work to the memory of my father and my brother. They were both immensely proud that I was working toward a PhD. I am sorry that they did not get to see my completed dissertation and I did not get to see them during my PhD program. It is with pride and affection that I dedicate this dissertation to them.

I would also like to dedicate this work to my mother. She never went to school but is immensely talented and well-mannered. She who is not able to read or write did her best so that all her children would be able to do both. She supported us to get the best possible education and I’m forever thankful for that.

–  –  –

I would like to express my deepest appreciation to my advisor, Dr. Nicole LaRonde. I was very fortunate to join her research group in May of 2012. She continually guided me in this journey and was very patient, supportive and motivating. I believe Nicole is one of the most inspiring female role models I’ve ever met in my life. She is very friendly, intelligent, determined, talented, honest, reliable, generous, patient and ambitious. She personally, trained me for most of the experiments I performed in the lab and provided insights to solve multiple research issues. Without her guidance and persistent help this dissertation would not have been possible.

I would also like to thank Dr. Tinoush Moulaei for sharing his knowledge with me and for providing guidance and help. I’m heavily indebted to him for so many encouraging and insightful discussions we had in the lab and I’m forever thankful for his support.





I would also like to take this opportunity and thank all members of the lab, past and present who provided help and made it fun working in the lab.

I would like to thank all the members of this thesis committee for their continued guidance support and especially for devoting so much time to get together in our second candidacy meeting. Dr. Rokita taught me a lot through his lectures on catalysis and through our meetings over the past years. He has certainly been the best teacher I’ve ever had in my life. I am grateful to Dr. Falvey and Dr.Sintim for their support and understanding. They were so kind and supportive to stay as members of my candidacy committee although I had to change majors from organic chemistry to biochemistry. Dr. Falvey devoted a lot of time to me, asking insightful questions during our meetings and has been very helpful and considerate in our meetings. Many thanks to Dr. sintim for being very responsive and helpful. I believe he

–  –  –

is a genius when it comes to organic chemistry and I’ve been very fortunate to be one of his students.

Finally, I am grateful to my entire old and new families: my parents, my brothers, and my sisters who stood by me and supported me in this effort. They encouraged me to continue to be successful and work hard to reach this goal. I have to thank my brother, Jaber for his support and love.

And Last, but certainly not the least, I have to thank Mehdi and his family. They have been very supportive of me and have always been by my side when I needed them. I have to thank you Mehdi for putting up with me throughout these years. You mean the world to me.

–  –  –

Table 4.1 Data collection and current refinement statistics for AMP bound Ct-Rio2 at the 78 pH of 5.

4.

Table 4.2 AMP-bound Ct-Rio2 Scalepack Logfile at pH 5.

4. The Ct-Rio2 crystals were 79 formed in the presence of ADP/BeCl2/KF in the hanging drop. The crystals were produced in 0.1M HEPES pH 7.0. 30% v/v jeffamine ED-2001 pH 7.0 at the 2:1 protein to well solution ratio.

Table 4.3 AMP-bound Ct-Rio2 phenix.

xtriage logfile at pH 5.4. The Ct-Rio2 crystals were 80 formed in the presence of ADP/BeCl2/KF in the hanging drop. The crystals were produced in 0.1M HEPES pH 7.0. 30% v/v jeffamine ED-2001 pH 7.0 at the 2:1 protein to well solution ratio.

Table 4.4 Data collection and current refinement statistics for AMP bound Ct-Rio2 at the 82 pH of 7.

The Ct-Rio2 crystals were formed in the presence of ADP/BeCl2/KF in the hanging drop. The crystals were produced in 0.1M HEPES pH 7.0. 30% v/v jeffamine ED-2001 pH

7.0 at the 2:1 protein to well solution ratio.

Table 4.5 AMP-bound Ct-Rio2 Scalepack Logfile at pH 7.

The Ct-Rio2 crystals were 83 formed in the presence of ADP/BeCl2/KF in the hanging drop. The crystals were produced in 0.1M HEPES pH 7.0. 30% v/v jeffamine ED-2001 pH 7.0 at the 2:1 protein to well solution ratio.

Table 4.6 AMP-bound Ct-Rio2 phenix.

xtriage logfile at pH 7. The Ct-Rio2 crystals were 84

–  –  –

formed in the presence of ADP/BeCl2/KF in the hanging drop. The crystals were produced in 0.1M HEPES pH 7.0. 30% v/v jeffamine ED-2001 pH 7.0 at the 2:1 protein to well solution ratio.

Table 4.7 Data collection and current refinement statistics for AMP-bound Ct-Rio2 at the 87 pH of 5.

4: The Ct-Rio2 crystals formed in the presence of ADP/Na2VO4 in the hanging drop at pH 5.4: The crystals were produced with 0.05M ammonium acetate, 0.01 magnesium chloride, 0.05M TRIS hydrochloride pH 7.5, 10% v/v 2-methyl-2,4-pentandiol Table 4.8 AMP-bound Ct-Rio2 phenix.xtriage logfile at the pH of 5.4: The Ct-Rio2 crystals 88 formed in the presence of ADP/Na2VO4 in the hanging drop at pH 5.4: The crystals were produced with 0.05M ammonium acetate, 0.01 magnesium chloride, 0.05M TRIS hydrochloride pH 7.5, 10% v/v 2-methyl-2,4-pentandiol.

Table 4.9 AMP-bound Ct-Rio2 Scalepack Logfile at pH 7.

The Ct-Rio2 crystals formed in 91 the presence of ADP/Na2VO4 in the hanging drop at pH 5.4: The crystals were produced in

0.05M ammonium acetate, 0.01 magnesium chloride, 0.05M TRIS hydrochloride pH 7.5, 10% v/v 2-methyl-2,4-pentandiol.

Figure 4.10 Crystal structure of Ct-Rio2 bound to ADP and transition state analog sodium 92 orthovanadate: The crystal structure of Ct-Rio2 shows a bilobal kinase domain (green and yellow) connected to an N-terminal winged helix domain (pink).

The “hinge” region connects the N- and C-terminal lobes of the kinase domain. The ADP binding loop “P-loop”, metal binding loop used to bind Mg2+, and catalytic loop which contains the catalytic D232 are indicated by arrows. The cartoon representation was made by PYMOL.

Table 6.1 Data collection and current refinement statistics for AMP-bound G228N.

The Ct- 114 Rio2 crystals formed in the presence of ATP in the hanging drop at pH 5.4.

–  –  –

Table 6.2 Data collection and current refinement statistics for AMP-bound G228A: The Ct- 115 Rio2 crystals formed in the presence of ATP in the hanging drop at pH 5.

4.

–  –  –

Figure 1.1 Mycobacterium tuberculosis and skin test: TB is an infectious disease caused 13 by Mtb.

According to the skin test results one third of the world population are infected by TB.

Figure 1.2 Global tuberculosis report: In 2014, 8.

7 million chronic cases of TB were 14 reported, leading to1.4 million deaths. Geographically, the burden of TB is highest in Asia and Africa. India and China together account for 40% of the world’s TB and Africa has 24% of the world’s TB infection.

Figure 1.3 TB pathophysiology: 1) Infection via inhalation of droplets; 2) Ingestion of 15 organisms by macrophages; 3) Multiplication within macrophages; 4) Minimal inflammatory response; 5) Extension of organisms into the bloodstream; 6) Hypersensitivity reaction; 7) Development of clinical infection.

Figure 1.4 The Mtb’s cell wall is consisted of several layers including phthiocerol 16 dimycocerosate (DIM/DIP) layer, mycolic acids, arabinogalactan (AG), and peptidoglycan, lipoarabinomannan, and phosphatidylinositol mannoside.

Figure 1.5 T7SS is encoded by ESX-1: ESX-1 is a specialized protein secretion system that 17 is encoded by genes of region of difference 1 (RD1) and its extended regions.

RD1 is comprised of nine genes (Rv3871 to Rv3879c), which are deleted from the primary cause of attenuation of the Mycobacterium bovis bacille Calmette Guérin (BCG) vaccine strain. That is attributed to a deletion of nine genes from the ESX-1 locus.

Figure 1.6 The representation of rRNA maturation: The 90S complex is cleaved on the 18 35S pre-rRNA at processing sites A0, A1 and A2, within the large 90S complex, to produce the pre-40S and the pre-60S particles.

The pre-40s particle is rapidly exported to the cytoplasm but the pre-60S particle remains in the nucleolus and undergoes a complex

–  –  –

maturation pathway in the nucleoplasm and is subsequently, delivered to the cytoplasm.

Figure 1.7 Formation of ribosomal beak: In the cytoplasm, the major structural 19 rearrangement that occurs on the pre-40S is the formation of the ribosomal “beak” structure that requires a cascade of phosphorylation and dephosphorylation events.

Cryo-EM and biochemical studies have revealed that the formation of the beak is concomitant with the release of assembly factors such as Ltv1 and Enp1.

Figure 1.8 Schematic representation of 40S subunit maturation in Saccharomyces 20 cerevisiae: Pre-40S particles at different stages of assembly are purified using TAP to obtain the systematic information about cytoplasmic synthetic pathway of maturation.

In the cytoplasm, the 20S pre-rRNA is first, methylated at the 3´ end of the 18S rRNA sequence by the function of Dim1.

Figure 1.9 Schematic representations of ribosomal protein and rRNA binding sites: In 21 the 40S structure model, the Rio2-binding site is located near the Rpss15.

Leger-Silvestre et al have reported that the pre-40S ribosome that lacks Rps15 fail to bind Rio2 and be exported to cytoplasm. In bacteria, the Rps18 and Rps16 homologues (S13 and S9) are phosphorylated at serine and threonine residue.

Figure 2.1 Schematic representation of EspB constructs: EspB constructs are designed 34 with an N-terminal 6x His tag followed by a TEV cleavage site.

Figure 2.2 Preparative gels of EspB292 after first His-trap column (A) and size 35 exclusion chromatography (B): Lane M is the protein ladder.

Gel of EspB292 displays pure protein at the expected molecular weight of 31.8 kDa.



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